Multiplex PCR Detection of Vibrio parahaemolyticus Virulence Factors
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Solution Overview
Problem
Current methods for detecting Vibrio parahaemolyticus, particularly those involving PCR, are not sensitive or specific enough to accurately identify all strains, including non-pathogenic ones, and lack internal controls to prevent false negatives, making them inefficient for timely and accurate detection of virulence factors like TDH and TRH.
Innovation Solution
A Taqman probe-based multiplex real-time PCR method that simultaneously detects Vibrio parahaemolyticus, TDH-related hemolysin, and thermostable direct hemolysin using specific primers and probes, along with an internal control to validate sample quality and prevent false negatives.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional PCR methods are used for detecting V. parahaemolyticus, then the detection process is simple, but the sensitivity and specificity are insufficient to accurately identify all strains including non-pathogenic ones
Solution Approach 1:
The patent combines multiple detection functions into a single multiplex PCR system that simultaneously detects V. parahaemolyticus species-specific genes (toxR), virulence factors (tdh, trh), and internal controls (E. coli yaiO gene) in one reaction, thereby improving detection sensitivity and specificity without proportionally increasing complexity
Solution Approach 2:
The detection method serves multiple functions simultaneously: species identification, virulence factor detection, and quality control validation, making the system universally applicable for comprehensive V. parahaemolyticus characterization while maintaining operational efficiency
2Reliability
If conventional PCR methods are used, then the procedure is straightforward, but there are no internal controls to prevent false negatives
Solution Approach 1:
The system incorporates an internal control (E. coli yaiO gene) that provides feedback on reaction quality and sample integrity, enabling real-time monitoring and validation of detection results to prevent false negatives while maintaining procedural simplicity
3Productivity
If multiple separate PCR reactions are performed to detect V. parahaemolyticus, TDH, and TRH, then each target can be detected specifically, but the detection time increases
Solution Approach 1:
The patent merges multiple detection targets (toxR, tdh, trh, and yaiO) into a single multiplex PCR reaction with optimized primer concentrations and fluorescent probes, achieving rapid simultaneous detection without sacrificing target-specific accuracy
4Adaptability or versatility
If conventional detection methods are used, then the cost is lower, but the ability to simultaneously detect multiple targets is limited
Solution Approach 1:
The multiplex PCR system achieves universal detection of multiple targets (species identification and virulence factors) in a single reaction, reducing overall reagent consumption and enabling comprehensive analysis without proportionally increasing costs
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method provides sensitive, specific, and rapid detection of Vibrio parahaemolyticus and its virulence factors in a single reaction, improving diagnostic accuracy and reducing the risk of false negatives by incorporating an internal control.
Implementation Method 1
contacting said sample with a plurality of pairs of primers, wherein the plurality of pairs of primer comprises: at least one pair of primers capable of hybridizing to the toxR gene of V. parahaemolyticus
Implementation Method 2
A Taqman probe-based multiplex real-time PCR method that simultaneously detects Vibrio parahaemolyticus, TDH-related hemolysin, and thermostable direct hemolysin
Data Source
AI summary
Methods and compositions for detection of V. parahaemolyticus, V. parahaemolyticus encoding TDH-related hemolysin, and V. parahaemolyticus encoding thermostable direct hemolysin are disclosed herein. In some embodiments, the presence or absence of V. parahaemolyticus, V. parahaemolyticus encoding TDH-related hemolysin, and/or V. parahaemolyticus encoding thermostable direct hemolysin in a sample is determined using multiplex nucleic acid-based testing methods.